Glucose dehydrogenases

ABSTRACT

Modified water-soluble glucose dehydrogenase having pyrrolo-quinoline quinone as a coenzyme are provided wherein at least one amino acid residue is replaced by another amino acid residue in a specific region. Modified water-soluble PQQGDHs of the present invention have improved thermal stability.

This application is a Continuation of co-pending application Ser. No.09/958,231 filed on Oct. 5, 2001. Application Ser. No. 09/958,231 is theU.S. National Phase under 35 U.S.C. § 371 of PCT InternationalApplication No. PCT/JP00/02322, filed Apr. 10, 2000, which designatedthe United States. This application claims priority of Application No.101143/1999 and 9152/2000 filed in Japan on Apr. 8, 1999 and Jan. 18,2000, respectively. Priority is claimed under 35 U.S.C. § 119 and § 120and the entire contents of all are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to the preparation of glucosedehydrogenases having pyrrolo-quinoline quinone as a coenzyme (PQQGDH)and their use for glucose assays.

BACKGROUND ART

Blood glucose is an important marker for diabetes. In the fermentativeproduction using microorganisms, glucose levels are assayed formonitoring the process. Conventional glucose assays were based onenzymatic methods using a glucose oxidase (GOD) or glucose-6-phosphatedehydrogenase (G6PDH). However, GOD-based assays required addition of acatalase or peroxidase to the assay system in order to quantitate thehydrogen peroxide generated by glucose oxidation reaction. G6PDHs havebeen used for spectrophotometric glucose assays, in which case acoenzyme NAD (P) had to be added to the reaction system.

An object of the present invention is to provide a modifiedwater-soluble PQQGDH with improved thermal stability.

DISCLOSURE OF THE INVENTION

We found that PQQGDHs with high stability are useful as novel enzymesalternative to the enzymes that have been used for enzymatic glucoseassays. PQQGDHs are useful as recognition elements of glucose sensorsbecause they have high oxidation activity for glucose and they arecoenzyme-bound enzymes that require no oxygen as an electron acceptor.

PQQGDHs catalyze the reaction in which glucose is oxidized to producegluconolactone. PQQGDHs include membrane-bound enzymes and water-solubleenzymes. Membrane-bound PQQGDHs are single peptide proteins having amolecular weight of about 87 kDa and widely found in variousgram-negative bacteria. Water-soluble PQQGDHs have been identified inseveral strains of Acinetobacter calcoaceticus (Biosci. Biotech.Biochem. (1995), 59(8), 1548-1555), and their structural genes werecloned to show the amino acid sequences (Mol. Gen. Genet. (1989),217:430-436). The water-soluble PQQGDH derived from A. calcoaceticus isa homodimer having a molecular weight of about 50 kDa.

Recently, a Dutch group made an X-ray crystal structure analysis of thewater-soluble PQQGDH to show the higher-order structure of the enzyme(J. Mol. Biol., 289, 319-333 (1999), The crystal structure of the apoform of the soluble quinoprotein glucose dehydrogenase fromAcinetobacter calcoaceticus reveals a novel internal conserved sequencerepeat; A. Oubrie et al., The EMBO Journal, 18(19) 5187-5194 (1999),Structure and mechanism of soluble quinoprotein glucose dehydrogenase,A. Oubrie et al., PNAS, 96(21), 11787-11791 (1999), Active-sitestructure of the soluble quinoprotein glucose dehydrogenase complexedwith methylhydrazine; A covalent cofactor-inhibitor complex, A. Oubrieet al.). These papers showed that the water-soluble PQQGDH is aβ-propeller protein composed of six W-motifs (FIG. 7).

As a result of careful studies to develop a modified PQQGDH that can beapplied to clinical tests or food analyses by improving the conventionalwater-soluble PQQGDH to increase the thermal stability, we succeeded inobtaining an enzyme with very high stability by introducing an aminoacid change into a specific region of the water-soluble PQQGDH.

Accordingly, the present invention provides a modified glucosedehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein anamino acid residue corresponding to serine 231 or glutamine 209 orglutamate 210 or aspartate 420 or alanine 421 in the water-solublePQQGDH derived from Acinetobacter calcoaceticus (hereinafter alsoreferred to as the wild-type PQQGDH) is replaced by another amino acidresidue. As used herein, the “modified glucose dehydrogenase” means aglucose dehydrogenase wherein at least one amino acid residue in anaturally occurring glucose dehydrogenase is replaced by another aminoacid residue. The amino acid numbering herein starts from the initiatormethionine as the +1 position.

The present invention also provides a modified glucose dehydrogenasehaving pyrrolo-quinoline quinone as a coenzyme wherein at least oneamino acid residue is replaced by another amino acid residue in one ormore regions selected from the group consisting of the regions definedby residues 48-53, 60-62, 69-71, 79-82, 91-101, 110-115, 127-135,147-150, 161-169, 177-179, 186-221, 227-244, 250-255, 261-263, 271-275,282-343, 349-377, 382-393, 400-403, 412-421, 427-432, 438-441 and449-468 in the amino acid sequence shown as SEQ ID NO: 1, characterizedin that it has higher thermal stability than that of the water-solublePQQGDH derived from Acinetobacter calcoaceticus. Preferably, themodified PQQGDH of the present invention has a residual activity that ishigher than the residual activity of the wild-type PQQGDH by 10% ormore, more preferably 20% or more, still more preferably 30% or moreafter heat treatment at 50° C. for 10 minutes. Preferably, the modifiedPQQGDH of the present invention has a heat inactivation half-life thatis longer than the heat inactivation half-life of the wild-type PQQGDHby 5 minutes or more, more preferably 15 minutes or more at 55° C. Inespecially preferred modified PQQGDHs of the present invention, at leastone amino acid residue is replaced by another amino acid residue in theregion defined by residues 227-244, 186-221 or 412-421 in the amino acidsequence shown as SEQ ID NO: 1. In still more preferred modified PQQGDHsof the present invention, serine 231 is replaced by an amino acidresidue selected from the group consisting of lysine, asparagine,aspartate, histidine, methionine, leucine and cysteine, or glutamine 209is replaced by lysine, or glutamate 210 is replaced by lysine, oraspartate 420 is replaced by lysine, or alanine 421 is replaced byaspartate in the amino acid sequence shown as SEQ ID NO: 1.

In another aspect, modified PQQGDHs of the present invention comprisethe sequence: Asn Leu Asp Gly Xaa231 Ile Pro Lys Asp Asn Pro Ser Phe AsnGly Val Val Ser (SEQ ID NO: 3) wherein Xaa231 represents a natural aminoacid residue other than Ser; or the sequence: (SEQ ID NO: 4) Gly Asp GlnGly Arg Asn Gln Leu Ala Tyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr ProThr Gln Xaa209 Xaa210 Leu Asn Gly Lys Asp Tyr His Thr Tyr Met Glywherein Xaa209 and Xaa210 represent any natural amino acid residue,provided that when Xaa209 represents Gln, Xaa 210 does not representGlu; or the sequence: Pro Thr Tyr Ser Thr Thr Tyr Asp Xaa420 Xaa421 (SEQID NO: 5) wherein Xaa420 and Xaa421 represent any natural amino acidresidue, provided that when Xaa420 represents Asp, Xaa421 does notrepresent Ala.

The present invention also provides a gene encoding any of the modifiedglucose dehydrogenases described above, a vector containing said geneand a transformant containing said gene, as well as a glucose assay kitand a glucose sensor comprising a modified glucose dehydrogenase of thepresent invention.

Enzyme proteins of modified PQQGDHs of the present invention have highthermal stability and high oxidation activity for glucose so that theycan be applied to highly sensitive and highly selective glucose assays.Especially, they are expected to provide the advantages that the enzymescan be produced at high yield with less inactivation duringpreparation/purification; the enzymes can be easily stored because oftheir high stability in solutions; the enzymes can be used to prepare anassay kit or an enzyme sensor with less inactivation; and the assay kitor enzyme sensor prepared with the enzymes has excellent storageproperties because of the high thermal stability.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the plasmid pGB2 used in the presentinvention.

FIG. 2 shows a scheme for preparing a mutant gene encoding a modifiedenzyme of the present invention.

FIG. 3 shows thermal stability of a modified enzyme of the presentinvention.

FIG. 4 shows substrate specificities of modified enzymes of the presentinvention.

FIG. 5 shows a glucose assay using a modified PQQGDH of the presentinvention.

FIG. 6 shows a calibration curve of an enzyme sensor using a modifiedPQQGDH of the present invention.

FIG. 7 shows the topology of a water-soluble GDH (Oubrie et al., FIG.4).

THE MOST PREFERRED EMBODIMENTS OF THE INVENTION

Structure of Modified PQQGDHs

We introduced random mutations into the coding region of the geneencoding the water-soluble PQQGDH by error-prone PCR to construct alibrary of water-soluble PQQGDHs carrying amino acid changes. Thesegenes were transformed into E. coli and screened for the residualactivity of the PQQGDHs after heat treatment to give a number of clonesthat express PQQGDHs with improved thermal stability.

Analysis of the nucleotide sequence of one of these clones showed thatSer 231 had been changed to Cys. When this amino acid residue wasreplaced by various other amino acid residues, mutant enzymes withhigher thermal stability than that of the wild type water-soluble PQQGDHwere obtained in every case.

The water-soluble PQQGDH has the structure of a β-propeller proteincomposed of six W-motifs. In the present invention, it was found thatthermal stability is improved by replacing Ser 231 in the loop regiondefined by residues 227-244 by another amino acid residue. Then,site-specific mutations were introduced into other loop regions to tryto improve the thermal stability. Mutant enzymes carrying Gln209Lys orGlu210Lys in the loop defined by residues 186-221 or Asp420Lys orAla421Asp in the loop defined by residues 412-421 showed improvedthermal stability.

Thus, it was demonstrated that water-soluble PQQGDHs with improvedthermal stability can be constructed by introducing a proper change intoa loop region according to the present invention. This is probablybecause the interaction between the loop regions connecting W-motifscontributes to the stabilization of the structure of the β-propellerprotein in water-soluble PQQGDHs. The residues Ser231, Gln209, Gly210,Asp420 and Ala421 shown above are only illustrative but not limiting thepresent invention. The present invention first showed in the art thatthermal stability of PQQGDHs can be improved by introducing a changeinto a specific site of the structural gene in a loop region, therebyproviding here a methodology for improving thermal stability of PQQGDHs.

Modified PQQGDHs of the present invention are characterized in that theycontain an amino acid residue change in a specific region in the aminoacid sequence of the wild-type PQQGDH shown as SEQ ID NO: 1.Accordingly, the present invention provides a modified glucosedehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein atleast one amino acid residue is replaced by another amino acid residuein one or more regions selected from the group consisting of the regionsdefined by residues 48-53, 60-62, 69-71, 79-82, 91-101, 110-115,127-135, 147-150, 161-169, 177-179, 186-221, 227-244, 250-255, 261-263,271-275, 282-343, 349-377, 382-393, 400-403, 412-421, 427-432, 438-441and 449-468 in the amino acid sequence shown as SEQ ID NO: 1.

In preferred modified PQQGDHs of the present invention, at least oneamino acid residue is replaced by another amino acid residue in theregion defined by residues 227-244, 186-221 or 412-421 in the amino acidsequence shown as SEQ ID NO: 1. In especially preferred modified PQQGDHsof the present invention, serine 231 is replaced by an amino acidresidue selected from the group consisting of lysine, asparagine,aspartate, histidine, methionine, leucine and cysteine, or glutamine 209is replaced by lysine, or glutamate 210 is replaced by lysine, oraspartate 420 is replaced by lysine, or alanine 421 is replaced byaspartate in the amino acid sequence shown as SEQ ID NO: 1.

In another aspect, modified PQQGDHs of the present invention comprisethe sequence: (SEQ ID NO: 3) Asn Leu Asp Gly Xaa231 Ile Pro Lys Asp AsnPro Ser Phe Asn Gly Val Val Ser

wherein Xaa231 represents a natural amino acid residue other than Ser;or the sequence: (SEQ ID NO: 4) Gly Asp Gln Gly Arg Asn Gln Leu Ala TyrLeu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr Gln Xaa209 Xaa210 LeuAsn Gly Lys Asp Tyr His Thr Tyr Met Glywherein Xaa209 and Xaa210 represent any natural amino acid residue,provided that when Xaa209 represents Gln, Xaa 210 does not representGlu; or the sequence: Pro Thr Tyr Ser Thr Thr Tyr Asp Xaa420 Xaa421 (SEQID NO: 5) wherein Xaa420 and Xaa421 represent any natural amino acidresidue, provided that when Xaa420 represents Asp, Xaa 421 does notrepresent Ala.

In modified glucose dehydrogenases of the present invention, other aminoacid residues may be partially deleted or substituted or other aminoacid residues may be added so far as glucose dehydrogenase activity isretained. Various techniques for such deletion, substitution or additionof amino acid residues are known in the art as described in Sambrook etal., “Molecular Cloning: A Laboratory Manual”, Second Edition, 1989,Cold Spring Harbor Laboratory Press, New York, for example. Thoseskilled in the art can readily test whether or not a glucosedehydrogenase containing such deletion, substitution or addition has adesired glucose dehydrogenase activity according to the teaching herein.Those skilled in the art can also predict a region having a loopstructure in water-soluble PQQGDHs derived from other bacteria accordingto the teaching herein and replace an amino acid residue in this regionto obtain modified glucose dehydrogenases with improved thermalstability. Particularly, an amino acid residue corresponding to serine231, glutamine 209, glutamate 210, aspartate 420 or alanine 421 in thewater-soluble PQQGDH derived from Acinetobacter calcoaceticus can bereadily identified by comparing the primary structures of proteins inalignment, so that modified glucose dehydrogenases can be obtained byreplacing such a residue by another amino acid residue according to thepresent invention. These modified glucose dehydrogenases are also withinthe scope of the present invention.

Process for Preparing Modified PQQGDHs

The sequence of the gene encoding the wild-type water-soluble PQQGDHderived from Acinetobacter calcoaceticus is defined by SEQ ID NO: 2.

Genes encoding modified PQQGDHs of the present invention can beconstructed by replacing the nucleotide sequence encoding an amino acidresidue occurring in a loop region as described above in the geneencoding the wild-type water-soluble PQQGDH by the nucleotide sequenceencoding an amino acid residue to be substituted. Various techniques forsuch site-specific nucleotide sequence substitution are known in the artas described in Sambrook et al., “Molecular Cloning: A LaboratoryManual”, Second Edition, 1989, Cold Spring Harbor Laboratory Press, NewYork, for example. Thus obtained mutant gene is inserted into a geneexpression vector (for example, a plasmid) and transformed into anappropriate host (for example, E. coli). A number of vector/host systemsfor expressing a foreign protein are known and various hosts such asbacteria, yeasts or cultured cells are suitable.

Random mutations are introduced by error-prone PCR into a target loopregion to construct a gene library of modified water-soluble PQQGDHscarrying mutations in the loop region. These genes are transformed intoE. Coli to screen each clone for the thermal stability of the PQQGDH.Water-soluble PQQGDHs are secreted into the periplasmic space when theyare expressed in E. coli, so that they can be easily assayed for enzymeactivity using the E. coli cells. This library is heated at 60-70° C.for about 30 minutes and then combined with glucose and a PMS-DCIP dyeto visually determine the residual PQQGDH activity so that clonesshowing residual activity even after heat treatment are selected andanalyzed for the nucleotide sequence to confirm the mutation.

Thus obtained transformed cells expressing modified PQQGDHs are culturedand harvested by centrifugation or other means from the culture medium,and then disrupted with a French press or osmotically shocked to releasethe periplasmic enzyme into the medium. The enzyme may beultracentrifuged to give a water-soluble PQQGDH-containing fraction.Alternatively, the expressed PQQGDH may be secreted into the medium byusing an appropriate host/vector system. The resulting water-solublefraction is purified by ion exchange chromatography, affinitychromatography, HPLC and the like to prepare a modified PQQGDH of thepresent invention.

Method for Assaying Enzyme Activity

PQQGDHs of the present invention associate with PQQ as a coenzyme incatalyzing the reaction in which glucose is oxidized to producegluconolactone.

The enzyme activity can be assayed by using the color-developingreaction of a redox dye to measure the amount of PQQ reduced withPQQGDH-catalyzed oxidation of glucose. Suitable color-developingreagents include PMS (phenazine methosulfate)-DCIP(2,6-dichlorophenolindophenol), potassium ferricyanide and ferrocene,for example.

Thermal Stability

Thermal stability of modified PQQGDHs of the present invention can beevaluated by incubating the enzyme of interest at a high temperature(for example, 55° C.), sampling aliquots at regular intervals andassaying the enzyme activity to monitor the decrease in the enzymeactivity with time. Typically, thermal stability of an enzyme isexpressed as a heat inactivation half-life, i.e. the time required forthe enzyme activity to be reduced to 50% (t_(1/2)). Alternatively,thermal stability can also be expressed as the residual enzyme activityafter heat treatment of the enzyme for a given period (the ratio of theactivity after heat treatment to the activity before heat treatment).

Modified PQQGDHs of the present invention are characterized by higherthermal stability than that of the wild-type PQQGDH. Thus, they have theadvantages that the enzymes can be produced at high yield with lessinactivation during preparation/purification; the enzymes can be easilystored because of their high stability in solutions; the enzymes can beused to prepare an assay kit or an enzyme sensor with less inactivation;and the assay kit or enzyme sensor prepared with the enzymes hasexcellent storage properties because of the high thermal stability.

Glucose Assay Kit

The present invention also relates to a glucose assay kit comprising amodified PQQGDH according to the present invention. The glucose assaykit of the present invention comprises a modified PQQGDH according tothe present invention in an amount enough for at least one run of assay.In addition to the modified PQQGDH according to the present invention,the kit typically comprises a necessary buffer for the assay, amediator, standard glucose solutions for preparing a calibration curveand instructions. Modified PQQGDHs according to the present inventioncan be provided in various forms such as freeze-dried reagents orsolutions in appropriate preservative solutions. Modified PQQGDHsaccording to the present invention are preferably provided in the formof a holoenzyme, though they may also be provided as an apoenzyme andconverted into a holoenzyme before use.

Glucose Sensor

The present invention also relates to a glucose sensor using a modifiedPQQGDH according to the present invention. Suitable electrodes includecarbon, gold, platinum and the like electrodes, on which an enzyme ofthe present invention is immobilized by using a crosslinking agent;encapsulation in a polymer matrix; coating with a dialysis membrane;using a photo-crosslinkable polymer, an electrically conductive polymeror a redox polymer; fixing the enzyme in a polymer or adsorbing it ontothe electrode with an electron mediator including ferrocene or itsderivatives; or any combination thereof. Modified PQQGDHs of the presentinvention are preferably immobilized in the form of a holoenzyme on anelectrode, though they may be immobilized as an apoenzyme and PQQ may beprovided as a separate layer or in a solution. Typically, modifiedPQQGDHs of the present invention are immobilized on a carbon electrodewith glutaraldehyde and then treated with an amine-containing reagent toblock glutaraldehyde.

Glucose levels can be measured as follows. PQQ, CaCl₂ and a mediator areadded to a thermostat cell containing a buffer and kept at a constanttemperature. Suitable mediators include, for example, potassiumferricyanide and phenazine methosulfate. An electrode on which amodified PQQGDH of the present invention has been immobilized is used asa working electrode in combination with a counter electrode (e.g. aplatinum electrode) and a reference electrode (e.g. an Ag/AgClelectrode). After a constant voltage is applied to the carbon electrodeto reach a steady current, a glucose-containing sample is added tomeasure the increase in current. The glucose level in the sample can becalculated from a calibration curve prepared with glucose solutions atstandard concentrations.

The disclosures of all the patents and documents cited herein areentirely incorporated herein as reference. The present applicationclaims priority based on Japanese Patent Applications Nos. 1999-101143and 2000-9152, the disclosure of which is entirely incorporated hereinas reference.

The following examples further illustrate the present invention without,however, limiting the same thereto.

EXAMPLE 1 Construction and Screening of a Mutant PQQGDH Gene Library

The plasmid pGB2 was obtained by inserting the structural gene encodingthe PQQGDH derived from Acinetobacter calcoaceticus into themulticloning site of the vector pTrc99A (Pharmacia) (FIG. 1). Thisplasmid was used as a template to introduce random mutations into thecoding region by error-prone PCR. The PCR reaction was carried out in asolution having the composition shown in Table 1 under the conditions of94° C. for 3 minutes, 30 cycles of 94° C. for 3 minutes, 50° C. for 2minutes and 72° C. for 2 minutes, and finally 72° C. for 10 minutes.TABLE 1 TaqDNA polymerase (5 U/μl) 0.5 μl Template DNA 1.0 μl Forwardprimer ABF 4.0 μl Reverse primer ABR 4.0 μl 10 × Taq polymerase buffer10.0 μl  1M β-mercaptoethanol 1.0 μl DMSO 10.0 μl  5 mM MnCl₂ 10.0 μl 10 mM dGTP 2.0 μl 2 mM dATP 2.0 μl 10 mM dCTP 2.0 μl 10 mM dTTP 2.0 μlH₂O 51.5 μl  100.0 μl 

The resulting mutant water-soluble PQQGDH library was transformed intoE. coli and each colony formed was transferred to a microtiter plate.After heating the plate at 60° C. for about 30 minutes, glucose andPMS-DCIP were added and the residual PQQGDH activity was visuallyevaluated. A number of clones showing PQQGDH activity even after heattreatment were obtained.

One of these clones was randomly selected and analyzed for thenucleotide sequence to show that serine 231 had been changed tocysteine.

EXAMPLE 2 Construction of Modified PQQGDH Genes

Based on the structural gene of the PQQGDH derived from Acinetobactercalcoaceticus shown as SEQ ID NO: 2, the nucleotide sequence encodingserine 231, glutamine 209, aspartate 420 or alanine 421 was replaced bythe nucleotide sequences encoding given amino acid residues bysite-directed mutagenesis according to a standard method as shown inFIG. 2 using the plasmid pGB2. Table 2 shows the sequences of thesynthetic oligonucleotide target primers used for mutagenesis. In Table2, “S231D” means that serine 231 is replaced by aspartate, for example.TABLE 2 (SEQ ID NOS: 6-16) S231D 5′-C CTT TGG AAT ATC TCC ATC AAG ATTTAA GC-3′ S231H 5′-C CTT TGG AAT ATG TCC ATC AAG ATT TAA GC-3′ S231K5′-C CTT TGG AAT TTT TCC ATC AAG ATT TAA GC-3′ S231L 5′-C CTT TGG AATCAT TCC ATC AAG ATT TAA GC-3′ S231M 5′-C CTT TGG AAT AGT TCC ATC AAG ATTTAA GC-3′ S231N 5′-C CTT TGG AAT ATT TCC ATC AAG ATT TAA GC-3′ I278F5′-C AAT GAG GTT GAA TTC ATC GTC AGA G-3′ Q209K 5′-C ACC ATT CAG TTC TTTTTG AGT TGG C-3′ E210K 5′-C ACC ATT CAG TTT TTG TTG AGT TGG C-3′ D420K5′-A CAT CGG TAC AGC TTT ATC ATA AGT AG-3′ A421D 5′-A CAT CGG TAC ATCGTC ATC ATA AGT AG-3′

A KpnI-HindIII fragment containing a part of the gene encoding thePQQGDH derived from Acinetobacter calcoaceticus was integrated into thevector plasmid pKF18k (Takara Shuzo Co., Ltd.) and used as a template.Fifty fmols of this template, 5 pmol of the selection primer attached tothe Mutan™-Express Km Kit (Takara Shuzo Co., Ltd.) and 50 pmol of thephosphorylated target primer were mixed with the annealing bufferattached to the kit in an amount equivalent to 1/10 of the total volume(20 μl), and the mixture was heated at 100° C. for 3 minutes to denaturethe plasmid into a single strand. The selection primer serves forreversion of dual amber mutations on the kanamycin-resistance gene ofpKF18k. The mixture was placed on ice for 5 minutes to anneal theprimers. To this mixture were added 3 μl of the extension bufferattached to the kit, 1 μl of T4 DNA ligase, 1 μl of T4 DNA polymeraseand 5 μl of sterilized water to synthesize a complementary strand.

The synthetic strand was transformed into a DNA mismatchrepair-deficient strain E. coli BMH71-18mutS and shake-culturedovernight to amplify the plasmid.

Then, the plasmid copies were extracted from the cultures andtransformed into E. coli MV1184 and then extracted from the colonies.These plasmids were sequenced to confirm the introduction of theintended mutations. These fragments were substituted for theKpnI-HindIII fragment of the gene encoding the wild-type PQQGDH on theplasmid pGB2A to construct genes for modified PQQGDHs.

EXAMPLE 3 Preparation of Modified Enzymes

The gene encoding the wild-type or each modified PQQGDH was insertedinto the multicloning site of an E. coli expression vector pTrc99A(Pharmacia), and the resulting plasmid was transformed into the E. colistrain DH5α. The transformant was shake-cultured at 37° C. overnight on450 ml of L medium (containing 50 μg/ml of ampicillin) in a Sakaguchiflask, and inoculated on 7 l of L medium containing 1 mM CaCl₂ and 500μM PQQ. About 3 hours after starting cultivation, isopropylthiogalactoside was added at a final concentration of 0.3 mM, andcultivation was further continued for 1.5 hours. The cultured cells wereharvested from the medium by centrifugation (5,000×g, 10 min, 4° C.),and washed twice with a 0.85% NaCl solution. The collected cells weredisrupted with a French press, and centrifuged (10,000×g, 15 min, 4° C.)to remove undisrupted cells. The supernatant was ultracentrifuged(160,500×g (40,000 r.p.m.), 90 min, 4° C.) to give a water-solublefraction, which was used in the subsequent examples as a crude enzymesample.

Thus obtained water-soluble fraction was further dialyzed against 10 mMphosphate buffer, pH 7.0 overnight. The dialyzed sample was adsorbed toa cation chromatographic column TSKgel CM-TOYOPEARL 650M (Tosoh Corp.),which had been equilibrated with 10 mM phosphate buffer, pH 7.0. Thiscolumn was washed with 750 ml of 10 mM phosphate buffer, pH 7.0 and thenthe enzyme was eluted with 10 mM phosphate buffer, pH 7.0 containing0-0.2 M NaCl at a flow rate of 5 ml/min. Fractions having GDH activitywere recovered and dialyzed against 10 mM MOPS-NAOH buffer, pH 7.0overnight. Thus, an electrophoretically homogeneous modified PQQGDHprotein was obtained. This was used in the subsequent examples as apurified enzyme sample.

EXAMPLE 4 Assay of Enzyme Activity

Enzyme activity was assayed by using PMS (phenazine methosulfate)-DCIP(2,6-dichlorophenolindophenol) in 10 mM MOPS-NaOH buffer (pH 7.0) tomonitor changes in the absorbance of DCIP at 600 nm with aspectrophotometer and expressing the reaction rate of the enzyme as therate of decrease in the absorbance. The enzyme activity for reducing 1μmol of DCIP in 1 minute was 1 U. The molar extinction coefficient ofDCIP at pH 7.0 was 16.3 mM⁻¹.

EXAMPLE 5 Evaluation of Thermal Stability of Crude Enzyme Samples

Each of the crude enzyme samples of the wild-type and modified PQQGDHsobtained in Example 3 was converted into a holoenzyme in the presence of1 μM PQQ and 1 mM CaCl₂ for 1 hour or longer and then incubated at 55°C. Aliquots were sampled at regular intervals and rapidly cooled on ice.These samples were assayed for the enzyme activity by the method ofExample 4 to determine the time required for reducing the activity to50% (t_(1/2)).

The results are shown in Table 3. TABLE 3 t_(1/2) (min) Wild type 10S231K 95 S231L 16 S231D 25 S231C 50 S231M 14 S231H 15 S231N 50 I278F 25Q209K 40 E210K 40 D420K 20 A421D 80

All the modified PQQGDHs of the present invention have a heatinactivation half-life at 55° C. longer than that of the wild-typePQQGDH, showing that they have higher thermal stability than that of thewild-type PQQGDH.

EXAMPLE 6 Evaluation of Thermal Stability of Purified Enzyme Samples

The purified samples of the wild-type enzyme and the modified enzymeS231K obtained in Example 3 were measured for the heat inactivationhalf-life at 55° C. in the same manner as in Example 5. The purifiedsamples of the wild-type enzyme and the modified enzyme S231K hadhalf-lives of 5 minutes and 41 minutes, respectively.

Then, each of the purified samples of the wild-type enzyme and themodified enzyme S231K obtained in Example 3 was converted into aholoenzyme in the presence of 1 μM PQQ and 1 mM CaCl₂ for 1 hour orlonger. Then, each sample was incubated at a given temperature in 10 mMMOPS buffer (pH 7.0) containing 1 μM PQQ and 1 mM CaCl₂ for 10 minutes,and then rapidly cooled on ice. These samples were assayed for theenzyme activity by the method of Example 4 to determine the residualactivity relative to the activity before heat treatment.

The results are shown in FIG. 3. The modified enzyme S231K had higheractivities than those of the wild-type enzyme at various temperatures of40-62.5° C.

EXAMPLE 7 Evaluation of Enzyme Activity

The crude enzyme sample of the modified enzyme S231K obtained in Example3 was converted into a holoenzyme in the presence of 1 μM PQQ and 1 mMCaCl₂ for 1 hour or longer. A 187 μl-aliquot was combined with 3 μl ofan activating reagent (prepared from 48 μl of 6 mM DCIP, 8 μl of 600 mMPMS and 16 μl of 10 mM phosphate buffer, pH 7.0) and 10 μl of glucosesolutions at various concentrations, and assayed for the enzyme activityat room temperature by the method shown in Example 4. The Km and Vmaxwere determined by plotting the substrate concentration vs. enzymeactivity. The S231K variant had a Km value for glucose of about 20 mMand a Vmax value of 3300 U/mg. The Km value of the wild-type PQQGDH forglucose reported to date was about 20 mM with the Vmax value being2500-7000 U/mg depending on the measurement conditions. These resultsshow that the modified PQQGDH S231K has high activity comparable to thatof the wild-type PQQGDH.

EXAMPLE 8 Evaluation of Substrate Specificity

Crude samples of various modified enzymes were tested for substratespecificity. The substrates tested were glucose, 2-deoxy-D-glucose,mannose, allose, 3-o-methyl-D-glucose, galactose, xylose, lactose andmaltose, and each sample was incubated with 20 mM of each substrate for30 minutes in the presence of 1 μM PQQ and 1 mM CaCl₂ and assayed forthe enzyme activity in the same manner as in Example 7 to determine therelative activity to the activity for glucose. As shown in FIG. 4, allthe modified enzymes of the present invention showed a similar substratespecificity to that of the wild-type enzyme.

EXAMPLE 9 Glucose Assay

A modified PQQGDH was used for assaying glucose. The modified enzymeS231K was converted into a holoenzyme in the presence of 1 μM PQQ and 1mM CaCl₂ for 1 hour or longer, and assayed for the enzyme activity inthe presence of glucose at various concentrations as well as 5 μM PQQand 10 mM CaCl₂ by the method described in Example 4 based on changes ofthe absorbance of DCIP at 600 nm. As shown in FIG. 5, the modifiedPQQGDH S231K can be used for assaying glucose in the range of 5 mM-50mM.

EXAMPLE 10 Preparation and Evaluation of an Enzyme Sensor

Five units of the modified enzyme S231K was freeze-dried with 20 mg ofcarbon paste. After thorough mixing, the mixture was applied only on thesurface of a carbon paste electrode preliminarily filled with about 40mg of carbon paste and polished on a filter paper. This electrode wastreated in 10 mM MOPS buffer (pH 7.0) containing 1% glutaraldehyde atroom temperature for 30 minutes followed by 10 mM MOPS buffer (pH 7.0)containing 20 mM lysine at room temperature for 20 minutes to blockglutaraldehyde. The electrode was equilibrated in 10 mM MOPS buffer (pH7.0) at room temperature for 1 hour or longer and then stored at 4° C.

Thus prepared enzyme sensor was used to measure glucose levels. FIG. 6shows the resulting calibration curve. Thus, the enzyme sensor having amodified PQQGDH of the present invention immobilized thereon could beused for assaying glucose in the range of 1 mM-12 mM.

INDUSTRIAL APPLICABILITY

Modified PQQGDHs of the present invention have excellent thermalstability so that they are expected to provide the advantages that theenzymes can be produced at high yield with less inactivation duringpreparation/purification; the enzymes can be easily stored because oftheir high stability in solutions; the enzymes can be used to prepare anassay kit or an enzyme sensor with less inactivation; and the assay kitor enzyme sensor prepared with the enzymes has excellent storageproperties because of the high thermal stability.

1. A modified glucose dehydrogenase having pyrrolo-quinoline quinone asa coenzyme wherein an amino acid residue corresponding to serine 231 inthe water-soluble PQQGDH derived from Acinetobacter calcoaceticus isreplaced by another amino acid residue.
 2. A modified glucosedehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein anamino acid residue corresponding to glutamine 209 in the water-solublePQQGDH derived from Acinetobacter calcoaceticus is replaced by anotheramino acid residue.
 3. A modified glucose dehydrogenase havingpyrrolo-quinoline quinone as a coenzyme wherein an amino acid residuecorresponding to glutamate 210 in the water-soluble PQQGDH derived fromAcinetobacter calcoaceticus is replaced by another amino acid residue.4. A modified glucose dehydrogenase having pyrrolo-quinoline quinone asa coenzyme wherein an amino acid residue corresponding to aspartate 420in the water-soluble PQQGDH derived from Acinetobacter calcoaceticus isreplaced by another amino acid residue.
 5. A modified glucosedehydrogenase having pyrrolo-quinoline quinone as a coenzyme wherein anamino acid residue corresponding to alanine 421 in the water-solublePQQGDH derived from Acinetobacter calcoaceticus is replaced by anotheramino acid residue.
 6. A modified glucose dehydrogenase havingpyrrolo-quinoline quinone as a coenzyme wherein at least one amino acidresidue is replaced by another amino acid residue in one or more regionsselected from the group consisting of the regions defined by residues48-53, 60-62, 69-71, 79-82, 91-101, 110-115, 127-135, 147-150, 161-169,177-179, 186-221, 227-244, 250-255, 261-263, 271-275, 282-343, 349-377,382-393, 400-403, 412-421, 427-432, 438-441 and 449-468 in the aminoacid sequence shown as SEQ ID NO: 1, characterized in that it has higherthermal stability than that of the water-soluble glucose dehydrogenasederived from Acinetobacter calcoaceticus.
 7. The modified glucosedehydrogenase of claim 3 wherein at least one amino acid residue isreplaced by another amino acid residue in the region defined by residues227-244 in the amino acid sequence shown as SEQ ID NO:
 1. 8. Themodified glucose dehydrogenase of claim 7 wherein serine 231 in theamino acid sequence shown as SEQ ID NO: 1 is replaced by another aminoacid residue.
 9. The modified glucose dehydrogenase of claim 3 whereinat least one amino acid residue is replaced by another amino acidresidue in the region defined by residues 186-221 in the amino acidsequence shown as SEQ ID NO:
 1. 10. The modified glucose dehydrogenaseof claim 9 wherein an amino acid residue corresponding to glutamine 209in the amino acid sequence shown as SEQ ID NO: 1 is replaced by anotheramino acid residue.
 11. The modified glucose dehydrogenase of claim 9wherein an amino acid residue corresponding to glutamate 210 in theamino acid sequence shown as SEQ ID NO: 1 is replaced by another aminoacid residue.
 12. The modified glucose dehydrogenase of claim 3 whereinat least one amino acid residue is replaced by another amino acidresidue in the region defined by residues 412-421 in the amino acidsequence shown as SEQ ID NO:
 1. 13. The modified glucose dehydrogenaseof claim 12 wherein an amino acid residue corresponding to aspartate 420in the amino acid sequence shown as SEQ ID NO: 1 is replaced by anotheramino acid residue.
 14. The modified glucose dehydrogenase of claim 12wherein an amino acid residue corresponding to alanine 421 in the aminoacid sequence shown as SEQ ID NO: 1 is replaced by another amino acidresidue.
 15. A glucose dehydrogenase having pyrrolo-quinoline quinone asa coenzyme comprising the sequence: Asn Leu Asp Gly Xaa23l Ile Pro LysAsp Asn Pro Ser Phe Asn Gly Val Val Ser

wherein Xaa231 represents a natural amino acid residue other than Ser.16. A glucose dehydrogenase having pyrrolo-quinoline quinone as acoenzyme comprising the sequence: Gly Asp Gln Gly Arg Asn Gln Leu AlaTyr Leu Phe Leu Pro Asn Gln Ala Gln His Thr Pro Thr Gln Xaa209 Xaa210Leu Asn Gly Lys Asp Tyr His Thr Tyr Met Gly

wherein Xaa209 and Xaa210 represent any natural amino acid residue,provided that when Xaa209 represents Gln, Xaa210 does not represent Glu.17. A glucose dehydrogenase having pyrrolo-quinoline quinone as acoenzyme comprising the sequence: Pro Thr Tyr Ser Thr Thr Tyr Asp Xaa420Xaa421

wherein Xaa420 and Xaa421 represent any natural amino acid residue,provided that when Xaa420 represents Asp, Xaa421 does not represent Ala.18. A gene encoding the modified glucose dehydrogenase of any one ofclaim
 1. 19. A vector comprising the gene of claim
 18. 20. Atransformant comprising the gene of claim
 18. 21. The transformant ofclaim 20 wherein the gene is integrated into the main chromosome.
 22. Aglucose assay kit comprising the modified glucose dehydrogenase of anyone of claim
 1. 23. A glucose sensor comprising the modified glucosedehydrogenase of any one of claim 1.